Translation is the endpoint of gene expression. Messenger RNAs are decoded using a complex machinery that has the ribosome as its centerpiece. Despite its importance, methods to track translation of cellular mRNAs remain undeveloped. Here, we build on years of method development in single-molecule translation to propose a novel, real-time method to track translation at codon resolution. Our approach uses fluorescently labeled tRNAs, ribosomes and other ligands to map translation start sites, coding sequences and termination sites. We build on our preliminary data demonstrating that real time single-molecule analysis of translation can be performed using fluorescently labeled tRNAs and ribosomes, harnessing recently-developed DNA sequencing instrumentation. We will focus our development efforts during the proposed funding period on three specific aims that will (1) create a collection of benchmarked fluorescent reagents for single-molecule translational profiling (2) use these reagents to characterize translational start sites, reading frames, termination sites and drug effects in three organisms: E. coli, yeast and human and (3) characterize rare translational phenomena, such as frameshifting, in these organisms. The goal of this work is provide real-time, genome-wide analysis of translational processes in these organisms. The endpoint of this proposal will be a core research facility that is dedicated to single-molecule translational analysis and providing access to the broader biomedical community. We believe these results will lead to a deeper understanding of the role of translational regulation in human disease.